流感病毒包膜蛋白M2免疫防護能力和防護機制研究
發(fā)布時間：2019-03-31 13:57
【摘要】： 流感的流行給人類健康帶來很大的危害,這就迫切要求開發(fā)出具有廣譜防護效果的流感疫苗。流感病毒包膜蛋白M2因其高度保守性而成為疫苗研制的重要靶蛋白。在本論文中,我們重點研究了流感病毒M2蛋白胞外區(qū)(M2e)的免疫防護能力和防護機制,并取得了以下創(chuàng)新性研究成果。 我們發(fā)現(xiàn)流感M2e具有很強的免疫原性,合成的M2e多肽無需載體蛋白就能誘導產(chǎn)生高滴度的特異性抗體,并且在攻毒實驗中能保護小鼠免于致死劑量流感病毒的攻擊。此外,研究發(fā)現(xiàn)M2e多肽能誘導產(chǎn)生特異性的T細胞反應(yīng),從而證明了在流感病毒M2e上存在Th細胞表位。這些研究成果部分闡明了流感病毒M2蛋白的免疫防護機制。 研究發(fā)現(xiàn)利用定點突變的方法將M2e多肽上的半胱氨酸殘基替換成絲氨酸后不會影響M2e多肽的抗原性,但會顯著降低其免疫原性,喪失誘導產(chǎn)生特異性免疫應(yīng)答的能力。此外,我們還發(fā)現(xiàn)M2e多肽可以通過半胱氨酸殘基形成的鏈間二硫鍵交聯(lián)形成多聚體。這表明M2e的半胱氨酸殘基對維持其免疫原性起到關(guān)鍵作用。 為了探討M2蛋白誘導的免疫應(yīng)答與病毒防護效果之間的關(guān)系,我們利用添加CpG-ODN佐劑的方法來影響M2e多肽誘導產(chǎn)生的免疫應(yīng)答。研究發(fā)現(xiàn)在M2e多肽中加入CpG-ODN能顯著提高其誘導產(chǎn)生的抗體水平和T細胞反應(yīng),并且促進免疫反應(yīng)向Th1型發(fā)展。但是免疫應(yīng)答的增強反而降低了高劑量病毒攻擊時的防護效果。 我們將M2e多肽與傳統(tǒng)的裂解疫苗聯(lián)合免疫,以增強裂解疫苗對不同亞型毒株的交叉防護能力。研究發(fā)現(xiàn)M2e多肽與裂解疫苗的聯(lián)合免疫不會影響血凝抑制抗體的產(chǎn)生,但是佐劑的使用對聯(lián)合免疫誘導的M2e特異性免疫應(yīng)答有重要影響。在鋁佐劑中,聯(lián)合免疫能誘導產(chǎn)生M2e特異性的免疫應(yīng)答,而在MF59佐劑中卻無此效果。此外,疫苗誘導的交叉防護效果也受所用佐劑的影響。在鋁佐劑中,加入M2e多肽能提高裂解疫苗的交叉防護效果,而在MF59佐劑中反而降低其交叉防護能力。這些研究結(jié)果提示M2實驗疫苗的防護效果也與佐劑的配伍相關(guān)。
[Abstract]:Influenza epidemic has brought great harm to human health, so it is urgent to develop influenza vaccine with broad-spectrum protective effect. Influenza virus envelope protein M2 has become an important target protein for vaccine development because of its high conservatism. In this paper, we focus on the immune protection ability and protection mechanism of influenza virus M2 protein extracellular domain (M2e), and obtain the following innovative research results. We found that influenza M2e has strong immunogenicity. The synthesized M2e peptide can induce high titer of specific antibody without vector protein, and can protect mice from lethal dose of influenza virus in the challenge experiment. In addition, it was found that M2e peptide could induce specific T cell response, which confirmed the existence of Th cell epitopes on influenza virus M2e. These results partly clarify the immune protection mechanism of influenza virus M2 protein. It was found that the substitution of cysteine residue from M _ 2e peptide with serine by site-directed mutation did not affect the antigenicity of M _ 2e peptide, but significantly decreased its immunogenicity and lost the ability to induce specific immune response. In addition, we also found that M _ 2e peptide can cross-link with disulfide bonds formed by cysteine residues to form polymers. This suggests that the cysteine residues of M2e play a key role in maintaining its immunogenicity. In order to investigate the relationship between the immune response induced by M2 protein and the protective effect of virus, we used the method of adding CpG-ODN adjuvant to influence the immune response induced by M2 peptide. It was found that the addition of CpG-ODN to M2e peptide could significantly increase the antibody level and T cell response induced by M2e peptide, and promote the development of immune response to Th1 type. However, the enhancement of immune response reduces the protective effect of high-dose virus attack. We combined the M2e peptide with the traditional lytic vaccine to enhance the cross-protection ability of the lytic vaccine against different subtypes of the vaccine. It was found that the co-immunization of M2e peptide and lytic vaccine did not affect the production of hemagglutination inhibition antibody, but the use of adjuvant had an important effect on the specific immune response of M2e induced by combined immunization. In aluminum adjuvant, co-immunization can induce M2e-specific immune response, but not in MF59 adjuvant. In addition, the cross-protective effect induced by the vaccine is also affected by the adjuvant used. In aluminum adjuvants, the addition of M _ 2e peptide could improve the cross-protection effect of lytic vaccine, while in MF59 adjuvant, the cross-protection ability of M2e peptide decreased. These results suggest that the protective effect of M2 experimental vaccine is also related to the compatibility of adjuvant.
【學位授予單位】：清華大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R392
本文編號：2450979
[Abstract]:Influenza epidemic has brought great harm to human health, so it is urgent to develop influenza vaccine with broad-spectrum protective effect. Influenza virus envelope protein M2 has become an important target protein for vaccine development because of its high conservatism. In this paper, we focus on the immune protection ability and protection mechanism of influenza virus M2 protein extracellular domain (M2e), and obtain the following innovative research results. We found that influenza M2e has strong immunogenicity. The synthesized M2e peptide can induce high titer of specific antibody without vector protein, and can protect mice from lethal dose of influenza virus in the challenge experiment. In addition, it was found that M2e peptide could induce specific T cell response, which confirmed the existence of Th cell epitopes on influenza virus M2e. These results partly clarify the immune protection mechanism of influenza virus M2 protein. It was found that the substitution of cysteine residue from M _ 2e peptide with serine by site-directed mutation did not affect the antigenicity of M _ 2e peptide, but significantly decreased its immunogenicity and lost the ability to induce specific immune response. In addition, we also found that M _ 2e peptide can cross-link with disulfide bonds formed by cysteine residues to form polymers. This suggests that the cysteine residues of M2e play a key role in maintaining its immunogenicity. In order to investigate the relationship between the immune response induced by M2 protein and the protective effect of virus, we used the method of adding CpG-ODN adjuvant to influence the immune response induced by M2 peptide. It was found that the addition of CpG-ODN to M2e peptide could significantly increase the antibody level and T cell response induced by M2e peptide, and promote the development of immune response to Th1 type. However, the enhancement of immune response reduces the protective effect of high-dose virus attack. We combined the M2e peptide with the traditional lytic vaccine to enhance the cross-protection ability of the lytic vaccine against different subtypes of the vaccine. It was found that the co-immunization of M2e peptide and lytic vaccine did not affect the production of hemagglutination inhibition antibody, but the use of adjuvant had an important effect on the specific immune response of M2e induced by combined immunization. In aluminum adjuvant, co-immunization can induce M2e-specific immune response, but not in MF59 adjuvant. In addition, the cross-protective effect induced by the vaccine is also affected by the adjuvant used. In aluminum adjuvants, the addition of M _ 2e peptide could improve the cross-protection effect of lytic vaccine, while in MF59 adjuvant, the cross-protection ability of M2e peptide decreased. These results suggest that the protective effect of M2 experimental vaccine is also related to the compatibility of adjuvant.
【學位授予單位】：清華大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R392
【引證文獻】
相關(guān)博士學位論文 前1條
1 范克偉;Itk信號轉(zhuǎn)導通路在A型流感病毒與T細胞相互作用中的功能研究[D];福建農(nóng)林大學;2012年
，本文編號：2450979
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